Aberrant phosphotyrosyl (pTyr)-dependent signaling is associated with the etiology of several cancers. For this reason, pharmacological agents are being developed to modulate pTyr-dependent cell signaling as potential new therapeutics. The approach used is predicated on the fact that pTyr-dependent signaling consists of three components related to cellular processing and manipulation of pTyr residues. These three components are: (1) the generation of pTyr-containing sequences by protein tyrosine kinases (PTKs), (2) the recognition and binding to pTyr-containing sequences by src homology 2 (SH2) domains, and (3) the destruction of pTyr sequences through phosphate ester hydrolysis by protein tyrosine phosphatases (PTPs). Accordingly, a unifying theme of this project is the design and synthesis of inhibitors directed at each of the three components. In the SH2 domain area, high affinity growth factor receptor-bound protein 2 (Grb2)-binding antagonists are being prepared as potential new therapeutics for erbB-2- and c-Met-dependent cancers. As part of a collaborative effort with clinical investigators (Drs. Don Bottaro and Marston Linehan, CCR, NCI), our Grb2 signaling inhibitors are being studied against von Hippel-Lindau (VHL)-dependent kidney cancers that rely on Grb2-dependent signaling pathways. In cellular studies, certain of these agents inhibit hepatocyte growth factor (HGF)-induced cell migration in Met containing fibroblasts at nanomolar concentrations and inhibit tubule formation potentially involved in angiogenesis. We have previously prepared a potent antagonist of Grb2 SH2 domain-binding that blocks growth factor-driven cell motility in vitro and angiogenesis in vivo. Our collaborators have now shown that this agent inhibits metastasis in vivo in two aggressive tumor models, without affecting primary tumor growth rate. This supports the potential efficacy of this compound in reducing the metastatic spread of primary solid tumors and establishes a critical role for Grb2 SH2 domain-mediated interactions in this process. We had previously reported using ring-closing metathesis (RCM) to prepare novel macrocycles designed as conformationally constrained peptide mimetics of our open-chain tripeptide inhibitors. The high synthetic complexity of these macrocyclic peptide mimetics was in part due to the stereoselective construction of a key upper ring-forming junction. In order to simplify synthesis, new macrocycle variants were prepared bearing upper ring junctions that: 1) were prepared non-stereoselectively, 2) utilized commercially available allylglycine residue, or 3) employed symmetrical achiral moieties. An extensive synthetic investigation has been undertaken within this macrocyclic family of peptide mimetics to identify structural features that enhance binding affinity. These investigations have advanced the field of macrocyclic peptidomimetic design and synthesis. Efforts were also undertaken to develop SH2 domain-directed peptide mimetic inhibitors of Shc-dependent signaling. Shc proteins are non-catalytic SH2 domain-containing docking modules that participate in a variety of cell regulatory processes associated with proliferation, survival, and apoptosis. Shc as well as Grb2 proteins are particularly important for downstream signaling of PTKs, where they have been shown to link activation of the cytoplasmic kinase domains with Ras effectors. Shc has also been shown to serve as a critical angiogenic switch for the production of vascular endothelial growth factor (VEGF) downstream from the c-Met and ErbB2 receptor tyrosine kinase (RTK) oncoproteins, where recruitment of Shc but not Grb2 has been shown to be a required event. Accordingly, disruption of Shc-dependent signaling through blockade of its SH2 domain interactions may afford a new therapeutic approach to cancers reliant on disregulation of such PTKs. It has previously been reported that the 14-mer zeta-chain-T cell receptor pY141 peptide, Ac-GHDGLpYQGLSTATK-amide binds to the Shc SH2 domain with an affinity of Kd = 50 uM. We prepared the hexamer partial sequence Ac-LpYQGLS-amide and found it to exhibit KD = 84 uM in a Biacore binding assay. Using a fluorescence anisotropy (FA) assay developed in the laboratory of Dr. Robert Fisher (SAIC-Frederick, Inc.) we undertook an exhaustive structure-activity study based on this hexamer sequence to determine the optimum placement of a fluoresceine group that would yield the highest affinity, and therefore the greatest sensitivity. We found that spacing between the fluoresceine and the peptide was critical. The highest affinity peptide gave a 100-fold enhancement (KD = 0.5 uM). Based on this peptide lead, non-fluoresceinated analogues were prepared containing functionality not present in genetically encoded amino acids, including N-alkylglycine (peptoid) residues. This work has resulted in the discovery of tetrameric peptide peptoid hybrids that exhibit low micromolar Shc SH2 domain binding affinity. Further structural optimization of these agents is in progress. In order to conduct cell-based studies, membrane carrier peptide sequences were chemically linked to select high affinity phosphopeptides resulting from these efforts. Preliminary data has indicated that these peptides can block the binding of Shc to activated Met in whole cells at low micromolar concentrations. In the phosphatase area, inhibitors are being developed against the YopH PTP, which is a pathogenic component of the potential bioterrosim agent Yersinia pestis. This work is being done in collaboration with Drs. Robert Ulrich (USAMRIID) and David Waugh (CCR, NCI). A focused library approach has been used wherein two aromatic fragments are joined together by a series of linker segments. This has led to the identification of low micromolar affinity inhibitors that are undergoing further optimization. A parallel approach to inhibitor development is being conducted based on the structural optimization of YopH substrates. Final inhibitors will be obtained by replacing the phosphate esters by hydrolytically stable bioisosteres. In the area of PTK inhibitor development, the high affinity Sugen Pharmaceuticals-derived c-Met kinase inhibitor SU11274 is being used as a platform to develop poly-valent kinase inhibitors. Based on molecular modeling (performed collaboratively within the Laboratory of Medicinal Chemistry by Dr. Marc Nicklaus), SU11274 is hypothesized to bind deep within the well-defined c-Met catalytic cleft. Linker chains are being appended onto the SU11274 core to allow binding within the catalytic cleft with inclusion of structural functionality exterior to catalytic cleft. One objective of this work is to join together multiple SU11274 units to take allow simultaneous binding to two or more c-Met kinase domains. This work has the potential to advance the development of PTK inhibitors in general.